The success of various synthetic nucleosides such as AZT, D4T, DDI, and DDC in inhibiting the replication of HIV in vivo or in vitro led researchers in the late 1980's to design and test nucleosides that substitute a heteroatom for the carbon atom at the 3′-position of the nucleoside. Norbeck, et al., disclosed that (±)-1-[cis-(2,4)-2-(hydroxymethyl)-4-dioxolanyl]thymine (referred to as (±)-dioxolane-T) exhibits a modest activity against HIV (EC50 of 20 uM in ATH8 cells), and is not toxic to uninfected control cells at a concentration of 200 uM. Tetrahedron Letters 30 (46), 6246, (1989). European Patent Application Publication No. 337 713 and U.S. Pat. No. 5,041,449, assigned to BioChem Pharma, Inc., disclose racemic 2-substituted-4-substituted-1,3-dioxolanes that exhibit antiviral activity. Published PCT application numbers PCT US91/09124 and PCT US93/08044 disclose isolated β-D-1,3-dioxolanyl nucleosides for the treatment of HIV infection. WO 94/09793 discloses the use of isolated β-D-1,3-dioxolanyl nucleosides for the treatment of HBV infection.
U.S. Pat. No. 5,047,407 and European Patent Application Publication No. 0 382 526, also assigned to BioChem Pharma, Inc., disclose that a number of racemic 2-substituted-5-substituted-1,3-oxathiolane nucleosides have antiviral activity, and specifically report that the racemic mixture of 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (referred to below as BCH-189) has approximately the same activity against HIV as AZT, with less toxicity. The (−)-enantiomer of BCH-189 (U.S. Pat. No. 5,539,116 to Liotta, et al.), known as 3TC, is now sold commercially for the treatment of HIV in humans in the United States. See also EP 513 200 B1.
It has also been disclosed that (−)-(cis)-FTC (4-amino-5-fluoro-1-(2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-2(1H)-pyrimidinone (2R-cis), or β-L-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane) has potent HIV activity. See Schinazi, et al., “Selective Inhibition of Human Immunodeficiency viruses by Racemates and Enantiomers of cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-Oxathiolane-5-yl]Cytosine” Antimicrobial Agents and Chemotherapy, November 1992, page 2423-2431. See also U.S. Pat. Nos. 5,814,639; 5,914,331; 5,210,085; U.S. Pat. No. 5,204,466, WO 91/11186, and WO 92/14743. The chemical structure of (−)-cis-FTC is shown below:

Because of the commercial importance of 1,3-oxathiolane nucleosides such as FTC, a number of processes for their production have been described in patents and scientific literature. The substituents on the chiral carbons (the specified purine or pyrimidine base (referred to as the C5 substituent)) and CH2OH (referred to as the C2 substituent)) of 1,3-oxathiolane nucleosides can be either cis (on the same side) or trans (on opposite sides) with respect to the oxathiolane ring system. Both the cis and trans racemates consist of a pair of optical isomers. Hence, each compound has four individual optical isomers. The four optical isomers are represented by the following configurations (when orienting the oxathiolane moiety in a horizontal plane such that the —S—CH2— moiety is in back): (1) cis (also referred to as β), with both groups “up”, which is the naturally occurring L-cis configuration (2) cis, with both groups “down”, which is the non-naturally occurring β-cis configuration; (3) trans (also referred to as the α-configuration) with the C2 substituent “up” and the C5 substituent “down”; and (4) trans with the C2 substituent “down” and the C5 substituent “up”. The two cis enantiomers together are referred to as a racemic mixture of β-enantiomers, and the two trans enantiomers are referred to as a racemic mixture of α-enantiomers. In general, it is fairly standard to be able to separate the pair of cis racemic optical isomers from the pair of trans racemic optical isomers. It is a significantly more difficult challenge to separate or otherwise obtain the individual enantiomers of the cis-configuration. For 3TC and FTC, the desired stereochemical configuration is the P-L-isomer.
The numbering scheme for the 1,3-oxathiolane ring in FTC is given below.
Routes to condense the 1,3-oxathiolane ring with a protected base
U.S. Pat. No. 5,204,466 discloses a method to condense a 1,3-oxathiolane with a protected pyrimidine base using tin chloride as a Lewis acid, which provides virtually complete β-stereoselectivity. See also Choi, et al, “In Situ Complexation Directs the Stereochemistry of N-Glycosylation in the synthesis of Oxathiolanyl and Dioxolanyl Nucleoside Analogues,” J. Am Chem. Soc. 1991, 213, 9377-9379. The use of tin chloride creates undesirable residues and side products during the reaction which are difficult to remove.
A number of U.S. patents disclose a process for the preparation of 1,3-oxathiolane nucleosides via the condensation of a 1,3-oxathiolane intermediate that has a chiral ester at the 2-position of the ring, with a protected base in the presence of a silicon-based Lewis acid. The ester at the 2-position must then be reduced to the corresponding hydroxymethyl group to afford the final product. See U.S. Pat. Nos. 5,663,320; 5,864,164; 5,693,787; 5,696,254; 5,744,596; and 5,756,706.
U.S. Pat. No. 5,763,606 discloses a process for producing predominantly cis-2-carboxylic or thiocarboxylic acid 1,3-oxathiolane nucleosides that includes coupling a desired, previously silylated purine or pyrimidine base with a bicyclic intermediate in the presence of a Lewis acid. U.S. Pat. No. 5,272,151 describes a process for the preparation of 1,3-dioxolane nucleosides that includes reacting a 2-O-protected-5-O-acylated-1,3-dioxolane with an oxygen- or nitrogen-protected purine or pyrimidine base in the presence of a titanium catalyst.
Choi, et al, “In Situ Complexation Directs the Stereochemistry of N-Glycosylation in the synthesis of Oxathiolanyl and Dioxolanyl Nucleoside Analogues,” J. Am Chem. Soc. 1991, 213, 9377-9379, reported that no coupling of the 1,3-oxathiolane with protected pyrimidine base occurs with HgCl2, Et2AlCl, or TiCl2(O-isopropyl)2 (see footnote 2). Choi also reported that the reaction between anomeric 1,3-oxathiolane acetates with silylated cytosine and virtually any common Lewis acid other than tin chloride resulted in the formation of inseparable mixtures of N-glycosylated anomers.
U.S. Pat. No. 5,922,867 discloses a method for preparing a dioxolane nucleoside that includes glycosylating a purine or pyrimidine base with a 2-protected-oxymethyl-4-halo-1,3-dioxolane.
U.S. Pat. Nos. 5,914,331, 5,700,937, 5,827,727, and 5,892,025, among others, to Liotta et al. describe coupling the 1,3-oxathiolanes disclosed therein with silyated 5-fluorocytosine in the presence of SnCl4 to form the β(−)isomer of FTC; and optionally removing the protecting groups.
Routes to Provide the 1,3-oxathiolane Nucleoside in the Desired Stereoconfiguration
Specific methods for preparing FTC in the desired stereoconfiguration in a substantially pure form are described in U.S. Pat. Nos. 5,914,331, 5,700,937, 5,827,727, and 5,892,025, among others, to Liotta et al. In one embodiment, the C5′-hydroxyl group of a mixture of nucleoside racemates is reacted with an acyl compound to form C5′-esters in which the nucleoside is in the “carbinol” end of the ester. The desired enantiomer can be isolated by treatment of the racemic mixture with an enzyme that hydrolyses the desired enantiomer (followed by extraction of the polar hydrolysate with a polar solvent) or by treatment with an enzyme that hydrolyses the undesired enantiomer (followed by removal of the undesired enantiomer with a polar solvent). Enzymes that catalyze the hydrolysis of 1,3-oxathiolane pyrimidine nucleosides include pig liver esterase, porcine pancreatic lipase, Amano PS-800 lipase, substillisin, and α-chymotrypsin.
Cytidine-deoxycytidine deaminase can be used to resolve racemic mixtures of 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane and its derivatives, including 2-hydroxymethyl-5-(5-fluoro-cytosin-1-yl)-1,3-oxathiolane. The enzyme catalyses the deamination of the cytosine moiety to a uridine. One of the enantiomers of 1,3-oxathiolane nucleosides is a preferred substrate for cytidine-deoxycytidine deaminase. The enantiomer that is not converted to a uridine (and therefore is still basic) is extracted from solution with an acidic solution. Cytidine-deoxycytidine deaminase can be isolated from rat liver or human liver, or expressed from recombinant sequences in a procaryotic system such as E. coli. 
Chiral chromatography can also be used to resolve cis-FTC enantiomers. For example, U.S. Pat. No. 5,892,025 to Liotta, et al. discloses a method for resolving a combination of the enantiomers of cis-FTC by passing the cis-FTC through an acetylated β-cyclodextrin chiral column.
Polymorphic Characterization
The ability of a compound to exist in different crystal structures is known as polymorphism. These different crystalline forms are known as “polymorphic modifications” or “polymorphs.” While polymorphs have the same chemical composition, they differ in packing and geometrical arrangement, and exhibit different physical properties such as melting point, shape, color, density, hardness, deformability, stability, dissolution, and the like. Depending on their temperature-stability relationship, two polymorphs may be either monotropic or enantiotropic. For a monotropic system, the relative stability between the two solid phases remains unchanged as the temperature is changed. In contrast, in an enantiotropic system there exists a transition temperature at which the stability of the two phases reverse. (Theory and Origin of Polymorphism in “Polymorphism in Pharmaceutical Solids” (1999) ISBN:)-8247-0237).
A number of compounds have been reported to exhibit polymorphism. As an early example; Gordon, et al. in U.S. Pat. No. 4,476,248, disclosed and claimed a new crystalline form of the drug ibuprofen as well as a process for producing it. The new crystalline form was reported to improve the manufacturability of ibuprofen. A structure more closely related to FTC, 3TC ((−)-cis-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidine-2-one; lamivudine), is also reported to exist in more than one crystalline form. Jozwiakowski, M. J., Nguyen, N. T., Sisco, J. M., Spancake, C. W. “Solubility Behavior of Lamivudine Crystal Forms in Recrystallization Solvents”, J. Pharm. Sci., 85, 2, p.193-199 (1996). See also U.S. Pat. No. 5,905,082 to Roberts et al., entitled “Crystalline Oxathiolane Derivatives,” issued May 18, 1999, and its PCT counterpart PCT/EP92/01213, describing two polymorphic forms of 3TC. Roberts et al. disclose that one polymorph is obtained when 3TC is crystallized from an aqueous solution. A second polymorph is obtained when 3TC is crystallized from non-aqueous media, or when the first form is melted and allowed to cool, or when the first form is ground or milled. Both polymorphic forms display-unique absorption bands, melting temperatures, and crystal energies.
(−)-cis-FTC produced by the above described methods has a distinct crystalline form, referred to herein as Form I (−)-cis-FTC. The angular positions (two theta) of the characteristic peaks in a powder X-ray diffraction pattern of (−) cis Form I FTC, shown in FIG. 7, are: 14.1°±0.1°, 19.9°±0.1°, 20.2°±0.1°, 20.6°±0.1°, 21.0°±0.1°, 22.4°±0.1°, 28.5°+0.1°, 29.5°±0.1°, and 32.6°±0.1°.
Additional polymorphs and other crystalline forms of FTC could have commercial value in manufacturing or other applications. It is therefore an objective of this invention to provide novel polymorphic and other crystalline forms of FTC.
It is another objective to provide novel methods for the preparation and isolation of polymorphic and other crystalline forms of FTC.
It is still another objective of the invention to provide therapeutic uses of FTC polymorphs and other phases of FTC.